The mechanism by which insulin regulates cell surface insulin receptors was examined in primary cultured rat adipocytes. When cells were incubated in insulin-free medium, specific 125I-insulin binding progressively increased over 3-4 days followed by a plateau of binding. Insulin prevented up-regulation at low doses (1 ng/ml), while higher doses (5-25 ng/ml) resulted in a net loss of surface receptors. A lag period of 4-6 h preceded insulin-induced changes in receptor number, and such a lag was seen prior to the inhibitory effect of insulin on the insertion of nascent receptors into the plasma membrane. Regulation of surface receptors continued after the removal of insulin, consistant with the idea that insulin generates a signal which can sustain receptor regulation in the absence of ligand. Thus, a 1-h insulin pulse (100 ng/ml) was sufficient to block up-regulation, whereas longer exposure times (4-12 h) produced a net loss of surface receptors. When cells were exposed to insulin for a fixed time (5 h), subsequent receptor loss was insulin dose-dependent. Thus, the net number of cell surface insulin receptors is determined by both insulin concentration and the duration of insulin exposure. Time course studies after a 12-h insulin pulse revealed a progressive loss of surface receptors for up to 36 h. At later times receptor number returned toward control values, thus demonstrating that triggering of receptor regulation is reversible. To determine whether insulin-induced down-regulation was mediated by receptor loss, or by receptor translocation to an intracellular site, we measured receptor distribution (cell surface and intracellular pools) in control and 72-h insulin-treated adipocytes. These data revealed that down-regulation was mediated by a net loss of receptors rather than by receptor redistribution. An early biological response of adipocytes to insulin is rapid endocytosis of insulin-receptor complexes, and we found that insulin plays an important role in regulating this endocytotic uptake rate. Thus, compared to freshly isolated cells, adipocytes cultured for 72 h in insulin-free medium had an enhanced ability to internalize both insulin receptors, whereas cells chronically treated with insulin endocytosed insulin-receptor complexes at a much slower rate. These data are interpreted as an insulin-mediated change in one of the cellular responses of adipocytes to the biological action of insulin.